Abstract Congenital Heart Disease (CHD), malformations of the heart present at birth, is the most common class of life-threatening birth defect. Atrioventricular Septal Defects (AVSDs) are a common and serious form of CHD in humans, comprising 5-10% of all CHD and a greater proportion of cases requiring surgical intervention. Gaining a mechanistic understanding of atrioventricular (AV) septation is an essential goal for improving clinical approaches to structural heart disease. This R01 is based on a paradigm shift in the understanding of AV septation. Whereas the canonical view of AV septation was based on intracardiac events centered at the endocardial cushion, recent work demonstrated that the T-box transcription factor Tbx5 is required in the Second Heart Field, outside of the heart proper, for AV septation. In this proposal, we harness this new understanding of AV septation to address the molecular and biochemical mechanisms underlying AV septation, using Tbx5 as an entry point. We propose to (1) define the molecular basis of TBX5 regulation of Wnt2, required for lung development and thereby AV septation, (2) identify the proteins that interact with and function with Tbx5 in the SHF for AV septation; and (3) define the biochemical mechanisms underlying the function of Tbx5 in the SHF. The ultimate aim of the proposed work is to understand the molecular and biochemical basis of AV septation. This proposal will increase understanding of the molecular ontogeny of human AVSDs. This work is an essential step towards defining the causes of human CHD and improving the natural history of patients with CHD.